The ability to eliminate posttransfusion hepatitis (PTH) and protect the blood supply worldwide depends upon the discovery and further characterization of the responsible virus agents. One of these agents, hepatitis B virus (HBV), is now eliminated from much of the blood supply by screening for the envelope antigenic determinants, or hepatitis B surface antigen (HBsAg), in serum. However, among three large prospective studies in which all blood samples were screened for HBsAg by radioimmunoassays, 0.3-1.7% of recipients of blood transfusions developed HBV, which accounts for 7-17% of PTH cases. These results demonstrate significant frequencies of PTH even with HBsAg screening. The association of antibodies against the hepatitis B core antigen (anti-HBc) with the transmission of HBV, and even more frequently with the transmission of non-A, non-B hepatitis (NANBH), suggests that HBV or a related agent present at low levels may contribute to PTH. The fact that only 2-9% of recipients of anti-HBc [+] blood develop posttransfusion hepatitis B, however, indicates that anti-HBc most often reflects past exposure and/or past infection. Rare cases of PTH have also been documented from blood with detectable antibodies against the hepatitis B surface antigen (anti-HBs), and studies have shown the presence of HBV DNA is serum samples from some anti- HBs [+] individuals by the highly sensitive polymerase chain reaction (Kaneko et al., PNAS (U.S.A) 86:312-316, 1989; Kaneko et al., Gastroenterol. 99:799-814, 1990). The finding of HBV DNA in HBsAg [-] cases of chronic liver disease underscores the need to find new ways to reliably detect and eliminate these types of HBV infections from the blood supply.
Another major cause of PTH is infection with HCV. Recently, serological assays have become available for the detection of HCV infections. Seroepidemiological data suggest that HCV accounts for the majority of PTH. It is estimated that roughly half of the patients infected with HCV develop chronic hepatitis, and that long term HCV infection is associated with the development of primary hepatocellular carcinoma (PHC). It has also been shown that one or more viral antibodies persist in patients who develop chronic infection. Many such patients also have persistent viremia, as shown by the detection of HCV RNA in serum by PCR (Wang et al., Gastroenterol, 103:609-616, 1992; Weiner et al., Lancet, 335:1-3, 1990; Bukh et al., PNAS (U.S.A), 89:187-191, 1992). Evidence is also accumulating that continued HCV replication is associated with the development and progression of chronic liver disease. Research is now being carried out worldwide to elucidate the natural history of infection and the markers which will be best suited for the screening of donor blood for the presence of HCV.
Relatively rare causes of vital hepatitis result from infection with one or more herpesviruses. Human cytomegalovirus (HCMV), for example, has been reported to be associated with neonatal hepatitis, in patients with PTH associated with liver transplantation, and in a few percent of patients with sporadic hepatitis. Epstein Bart virus (EBV) has been found among patients with hepatitis following liver transplantation. Herpes simplex virus (HSV) is associated with hepatitis among immunosuppressed bone marrow recipients, even when the transplantation is autologous. Associations have also been shown between HSV and fulminant neonatal hepatitis and in acute hepatitis in adults. Despite the fact that herpesvirus associated hepatitis is rare, these vital etiologies need to be considered in blood screening when making a differential diagnosis of hepatitis.
The problems of PTH associated with HBV infection have been solved among the subset of potential blood donors who are asymptomatic but have detectable HBsAg in blood by current screening procedures. Clearly, a hallmark of chronic HBV infection is the variability in pathogenesis which accompanies such infections. Most people who become acutely infected with HBV resolve virus infection without the development of HBsAg, virus, or hepatitis. A small percentage develop fulminant hepatitis. Others develop acute hepatitis, followed by resolution in which HBsAg and HBV particles are cleared from the blood, followed by seroconversion to anti-HBs. Still others do not clear virus, but instead develop the chronic carrier state, which may be asymptomatic or may result in the appearance and progression of chronic hepatitis, cirrhosis, and eventually PHC. Among the more than 300 million estimated HBV carriers worldwide, at least 250,000 new cases of PHC are diagnosed annually; less than 3% of these patients survive 5 years. Given that the relative risk of HBV carriers developing PHC is more than 200:1, and that there are few treatments available for liver diseases associated with chronic HBV infection, including PHC, there is a real need to characterize HBV in HBsAg [-] infections from both the standpoints of screening and management.
While it has been suggested that the nature of the cellular and humoral immune responses against HBV are likely to be important to the outcome of acute infection, variation in the virus may also be very important to the host-virus relationship that evolves. To place this in context, while there are more than 300 million HBV carriers worldwide, it is estimated that about half of the world's population is infected with HBV but does not develop the HBsAg [+] carrier state.
U.S. Pat. No. 4,777,240 (Moriarty et al.) discloses methods for assaying for the presence of HBxAg and anti-HBx employing antigenic synthetic polypeptides. Preferred polypeptides have the following sequences:
Leu Ser Ala Met Ser Thr Asp Leu Glu Ala Tyr Phe Lys Asp (SEQ ID NO: 1); PA1 Leu Phe Lys Asp Trp Glu Leu Gly Glu Ile Arg Leu Lys Val (SEQ ID NO: 2); and
Ala Pro Ala Pro Cys Asn Phe Thr Set Ala (SEQ ID NO: 3). Antibodies capable of reacting with one of the aforementioned antigenic polypeptides and a diagnostic assay system for determining the presence of a detectable amount of HBxAg in a body sample are also disclosed.
Assay systems are described. Such a system can include a first reagent containing receptor molecules that include an antibody combining site (such as antibodies) which is capable of reacting with a synthetic polypeptide of the invention. Indicating means such as a fluorescent dye, a radioactive element, or an enzyme-linked antibody raised to the first reagent's receptors are also provided. Such a reagent system is said to be useful for enzyme-linked immunosorbant assay (ELISA). Diagnostic assays employing anti-polypeptide receptor are also disclosed.
Methods for assaying for the presence of a detectable amount of HBxAg by admixing proteins from a body sample to be assayed with antibodies (receptors) capable of reacting with a synthetic polypeptide of the invention are also provided. The antibody may be labeled (radioactive or enzyme label) to signal an immunoreaction between HBxAg and the receptors. In these methods, the body sample is preferably bound to a solid matrix before being mixed with the receptors. The label may be a separate molecule or may be a part of the receptor.
These assay methods and systems are said to be useful for identifying the X protein in an ELISA or Western blot format.
In one example, serum from a patient diagnosed as having a hepatocellular carcinoma was found to contain antibodies that bound to one of the polypeptides of the invention.
Feitelson et al., J. Hepatology13:S58-S60 (1991) showed HBV DNA in serum from HBsAg negative renal dialysis patients by polymerase chain reaction (PCR) employing the following primers: MF03 (residues 1903-1929) and MF04 (residues 2436-2412) for amplification of the core region; MF24 (residues 1231-1247) and MF04 for amplification of the X plus core region; MF16 (residues 1929-1903; complementary to MF03) and MF24, for amplification of the X region; MF06 (residues 2850-2873) and MF07 (residues 154-132) for amplification of the preS region. The HBV-DNA and clone numbering system is used to designate the residues. The clone used was ayw; position 1 is at the Eco R1 site in HBV DNA (Tiollars, P., C. Pourcel, A. Dejean, 1985Nature, 317:489-495).
It was found that there detectable X deletion mutants months or years before the appearance of HBsAg and wild-type HBV-DNA in serum, suggesting that these mutant strains could infect liver and replicate at low levels relative to wild type. Virus particles carrying different X region deletion mutations were found in a single infection. Results also indicated that patients could be infected with different X deletion mutants while on dialysis and/or that two or more mutants may undergo genetic recombination.
Similar results in WHV-infected woodchucks are also discussed. Among infected individuals in which only X region deletion mutants are present in serum, most with detectable X antigen in serum are also positive for surface antigen, anti-core, and virus DNA. In contrast, those without evidence of X polypeptide production are usually negative for surface antigen, anti-core and virus DNA by conventional techniques. Some in the latter group have detectable anti-pol as the only serological marker of infection.
Perhaps the most challenging problem in HBV associated PTH is to find ways of detecting HBV transmission in blood lacking all serological markers of infection. This is important because there is growing evidence that HBV transmitted in blood lacking all conventional markers of infection. It has been shown that HBV DNA sequences are detectable in the serum (Theirs et al., Lancet ii:1273-1276, 1988; Wang et al., J. Infect. Diseases, 163:397-399, 1991) and liver (Figus et al., Hepatology, 4:364-368, 1984; Paterlini et al., N. Engl. J. Med., 323:80-85, 1990) of patients without other HBV markers. An anti-HBs-like monoclonal antibody has been developed to detect such agents by immunoprecipitation. The agent binding to this antibody has been shown to transmit hepatitis in chimpanzees (Wands et al., PNAS (U.S.A.), 79:7552-7556, 1982). However, there remains a need for methods capable of detecting active HBV infection, characterizes by virus replication, in HBsAg [-], anti-HCV [-] PTH.